Pilot evaluation of pathogen isolation using targeted amplicon next-generation sequencing on sonication fluid for the diagnosis of periprosthetic joint infections

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Abstract Background Periprosthetic joint infection (PJI) remains difficult to diagnose due to the limited sensitivity of conventional culture, which frequently yields false-negative results. Targeted amplicon next-generation sequencing (tNGS) offers a method capable of identifying fastidious organisms, polymicrobial infections, and antimicrobial resistance genes. This pilot proof-of-concept study evaluated the feasibility of applying tNGS to sonicated fluid from orthopedic implants compared with conventional culture. Methods Six sonicated fluid samples were collected from patients undergoing revision arthroplasty or removal of osteosynthesis devices between August and September 2024. Implants were processed in saline or Ringer’s lactate and subjected to a standardized sonication protocol, followed by aerobic and anaerobic culture. Parallel tNGS analysis targeted bacterial 16S rRNA and fungal internal transcribed spacer regions, sequenced on an Illumina MiSeq platform with bioinformatic quality control and contaminant filtering. Microbial identifications and predicted resistance genes were compared descriptively across methods. Results Patients ranged in age from 23 to 76 years; comorbidities included diabetes, dyslipidemia, and hypertension. Conventional culture identified pathogens in four of six cases but missed fungi, several bacterial species, and resistance markers. In contrast, NGS successfully processed all six sonicated fluid samples, confirming the technical feasibility of the approach while confirming culture-positive organisms such as Staphylococcus aureus and detecting additional clinically relevant bacteria, fungi ( Cladosporium , Rhodotorula ), and resistance genes ( mecA , aph3′ ) that culture failed to identify. Two samples were negative by both methods. Discrepancies were more frequent in samples processed with Ringer’s lactate, suggesting interference with sequencing. Conclusions This pilot study demonstrated the feasibility of applying targeted amplicon next-generation sequencing to sonicated fluid from orthopedic implants and highlighted its potential to complement conventional culture by identifying fastidious organisms, polymicrobial infections, and resistance determinants. These findings highlight its potential to improve diagnostic accuracy and guide personalized antimicrobial therapy in PJI. Although the small sample size and descriptive design limit generalizability, pilot study provides early evidence supporting the feasibility of applying tNGS to sonicated fluid and identifies methodological considerations for future research. Larger prospective trials are warranted to validate these results and establish NGS as an adjunct or alternative to culture in orthopedic infection diagnostics. Trial registration The study protocol was approved by the local Institutional Review Board (CAAE: 46277421.1.0000.5479; approval number: 4.713.409).